Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2025.05.13.653678

Examination of the enrichment of neuronal extracellular vesicles from cell conditioned media and human plasma using an anti-NCAM immunocapture bead approach

Collier, M. E. W.·Allcock, N.·Sylvius, N.·Cassidy, J.·Giorgini, F.
View original source ↗Export metadata ↓

Abstract

BackgroundThe isolation of neuron-derived extracellular vesicles (nEVs) from biofluids offers the potential to discover novel biomarkers to aid in diagnosis and treatment of psychiatric and neurodegenerative diseases. A few studies have used anti-NCAM antibody-bead-based immunocapture to enrich nEVs from plasma, some with little method validation. We therefore examined in detail this method for nEV enrichment. MethodsEVs were isolated from SH-SY5Y cell-conditioned media by precipitation, or from plasma using size exclusion chromatography. EVs were characterised using nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and immunoblot analysis. SH-SY5Y-EVs were incubated with anti-NCAM immunocapture beads and examined by flow cytometry, immunoblot analysis and scanning electron microscopy (SEM). Immunocaptured plasma-derived EVs were examined using a sensitive NCAM ELISA, SEM and qPCR for miRNAs. ResultsCharacterisation of SH-SY5Y-derived and plasma-derived EVs revealed the expected size distributions of EVs using NTA, the presence of EV markers using immunoblot analysis, and a cup-shaped morphology using TEM. Anti-NCAM beads, but not anti-L1CAM or IgG beads, captured NCAM-positive SH-SY5Y-EVs as shown by flow cytometry and immunoblot analysis. Both SH-SY5Y and plasma-derived EVs were visualised on the surface of anti-NCAM immunocapture beads using SEM. A sensitive NCAM ELISA detected NCAM antigen in plasma-derived EVs immunocaptured on anti-NCAM beads. qPCR analysis of plasma-derived EVs detected many miRNAs in total plasma-EVs with high expression of hsa-miR-16-5p, hsa-miR-451a and hsa-miR-126-3p. However, only between two and seven miRNAs were detected in EVs captured on anti-NCAM-beads from three blood donors. Finally, tissue distribution analysis of miRNAs from plasma-derived EVs on anti-NCAM beads revealed that these miRNAs are enriched in tissues or organs such as blood vessels, lung, bone, thyroid and heart, but were not enriched for brain-derived miRNAs. Discussion: This study indicates that anti-NCAM beads can efficiently enrich NCAM-positive EVs from cell culture conditioned media. However, nEV levels in small volumes of plasma are possibly too low to enable efficient anti-NCAM immunocapture for subsequent miRNA analysis. Other neuron-specific markers with high expression levels on nEVs are therefore required for processing patient samples where plasma volumes are likely to be low, and to allow efficient isolation of nEVs in clinical studies for subsequent cargo analysis.

Source connections

Explore related subjects

cell biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Collier, M. E. W., Allcock, N., Sylvius, N., Cassidy, J., Giorgini, F.. 2025-05-14. Examination of the enrichment of neuronal extracellular vesicles from cell conditioned media and human plasma using an anti-NCAM immunocapture bead approach. https://doi.org/10.1101/2025.05.13.653678

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

The nuclear membrane protein Samp1 links peripheral genome organization to the myogenic transcriptional program

Samp1 is an inner nuclear membrane protein required for myogenic differentiation and involved in chromatin organization at the nuclear periphery. Here, we investigated whether these functions are connected by studying the effects of Samp1 depletion during C2C12 myogenic differentiation using immunofluorescence microscopy, RNA sequencing, FRIC, and chromosome-positioning analysis. Samp1-depleted cells showed strongly reduced MyHC expression and virtually abrogated multinucleated fiber formation. Although cell-cycle withdrawal was not prevented, the transcriptional program driving differentiation was drastically perturbed, with reduced muscle-associated transcripts and incomplete repression of genes normally downregulated during myogenesis. Samp1 depletion also disrupted peripheral chromatin organization and prevented the accumulation of peripheral heterochromatin typically seen during differentiation. In addition, radial chromosome distribution was disrupted, evidenced by the failure of chromosome 8 to reposition to the nuclear periphery during differentiation. Together, these findings link the requirement for Samp1 in myogenic differentiation to its role in genome organization at the nuclear periphery.

cell biology↗
bioRxiv2026

Unraveling the metabolic landscape of alkaptonuria through a human-relevant in vitro liver disease model

Alkaptonuria (AKU) is a rare inherited metabolic disorder of tyrosine catabolism caused by a deficient homogentisate 1,2-dioxygenase (HGD) enzyme. This results in the accumulation of homogentisic acid (HGA), driving a progressive multisystem pathology characterized by debilitating early-onset osteoarthritis due to connective tissue degeneration. While previous in vitro studies have primarily relied on exogenous HGA exposure in osteoarticular cell models, the direct metabolic consequences of endogenous HGD deficiency within its native hepatic context remain poorly understood. Here, we established the first human-relevant HGD knockout hepatic in vitro model using a universal in-house-developed homology-directed repair approach. Integrative multi-omic analysis revealed that HGD deficiency induces widespread metabolic rewiring extending beyond disrupted tyrosine catabolism. HGD-deficient hepatocytes exhibited elevated oxidative stress accompanied by impaired mitochondrial respiration and a pseudohypoxic metabolic adaptation toward increased glycolytic dependency. Despite this glycolytic shift, the cells displayed reduced anabolic and translational activity alongside attenuated proliferation, consistent with a chronic stress-adaptive survival state rather than a proliferative metabolic phenotype. This study provides systems-level insights into the pathophysiology of AKU and establishes a versatile platform for mechanistic and therapeutic investigation.

cell biology↗
bioRxiv2026

The circadian clock regulates KCNH2 (hERG) promoter activity through daily temperature rhythms.

Background: KCNH2 encodes Kv11.1 channel proteins that conduct the rapidly activating delayed-rectifier K+ current (IKr), which is critical for cardiac repolarization. KCNH2 encodes two functional isoforms, Kv11.1a and Kv11.1b, via alternative transcription start sites. Kv11.1a is the principal determinant of cardiac IKr and ventricular repolarization. The circadian clock, a transcriptional-translational feedback loop that cycles with a period of ~24 hours and drives the circadian expression of many genes, including Kcnh2 in the mouse heart. Because daily body temperature rhythms provide a systemic signal that synchronizes cardiac circadian clocks, we tested whether physiological temperature cycles drive the circadian promoter activity of the cloned human KCNH2 (hKCNH2) promoter. Hypothesis: hKCNH2 is a direct transcriptional target of the circadian clock, with temperature driving its promoter activity through BMAL1:CLOCK acting at a conserved tandem E-box. Methods: We cloned the conserved proximal promoter of KCNH2 (-1631 bp upstream of Kv11.1a exon 1) to generate hKCNH2 promoter luciferase reporter constructs. Constructs were transfected into C2C12 myotubes and synchronized by serum shock (static 37{degrees}C) or temperature cycling (36.5-38.5{degrees}C). Bioluminescence was recorded and assessed for period, phase, and amplitude. BMAL1:CLOCK dependence was tested via dominant-negative CLOCK{Delta}19 co-expression. Results: Temperature cycling did not exhibit the rapid damping characteristic of serum-shock-synchronized oscillations, consistent with continuous entrainment by an external zeitgeber rather than a free-running oscillator. Deletion analysis identified a conserved tandem E-box required for oscillation under both serum shock and temperature cycling, and for BMAL1:CLOCK-dependent transactivation (1.75 {+/-} 0.21 vs. 0.86 {+/-} 0.06 RLU, p = 0.0038). CLOCK{Delta}19 reduced hKCNH2 promoter amplitude under temperature cycling without altering period. Conclusion: The circadian clock regulates KCNH2 promoter activity through daily temperature rhythms.

cell biology↗